US8242242B2 - Preparation of an artificial transcription factor comprising zinc finger protein and transcription factor of prokaryote, and a use thereof - Google Patents
Preparation of an artificial transcription factor comprising zinc finger protein and transcription factor of prokaryote, and a use thereof Download PDFInfo
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- US8242242B2 US8242242B2 US12/444,842 US44484206A US8242242B2 US 8242242 B2 US8242242 B2 US 8242242B2 US 44484206 A US44484206 A US 44484206A US 8242242 B2 US8242242 B2 US 8242242B2
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
Definitions
- the present invention relates to artificial transcription factors capable of artificially regulating gene expression of Escherichia coli by using zinc finger proteins and transcription factors of prokaryote, and engineered E. coli using the same.
- the zinc fingers are known as a DNA binding motif of a DNA binding proteins that are most frequently discovered in eukaryote.
- the zinc fingers are an active domain that can recognize sequence-specifically a target sequence and can work as transcription repressor by themselves.
- a new transcription factor can be prepared by fusing the zinc finger proteins with a transcription activating (or suppressing) domain, wherein the zinc finger proteins is used as a DNA binding domain.
- An object of the present invention is to provide a preparation for artificial transcription factors that can artificially up- or down-regulate the gene expression by fusing zinc finger domains to transcription factors of prokaryotes as an effector domain, and also provide an engineered E. coli having various phenotype specificities.
- an effector domain a transcription factor of prokaryotes used as an effector domain.
- Said transcription factor of prokaryotes used as an effector domain may be CRPs (Catabolite regulatory proteins) (or cyclic AMP receptor proteins) or its derivatives, such as a wild type CRP (CRP W, residue 1-209), CRP Del 137 (residue 137-190), or CRP Del 180 (residue 1-180).
- the zinc finger domains may be identified from human genes and selected from the group consisting of nucleic acid sequences having SEQ ID NOs. 13 to 64.
- the present invention provides a reporter plasmid in which the gene encoding enhanced green fluorescent protein (EGFP) was used as a reporter to select the most potent effector domains.
- EGFP enhanced green fluorescent protein
- Two different reporter plasmids were constructed by inserting the target DNA sequences for the artificial transcription factors into two different parts of the reporter gene. Specifically, the resulting plasmids were pEGFP-A, which contained the artificial transcription factor target sequence upstream of the reporter promoter ( ⁇ 67 to ⁇ 50), and pEGFP-R, which contained the artificial transcription factor target sequence downstream of the promoter (+24 to +41).
- the present invention provides an engineered E. coli having various phenotypes by introducing the artificial transcription factor thereto, such as E. coli having the resistance to heat or cold shock, E. coli having the improvement of growth, and E. coli having the resistance to osmotic pressure.
- E. coli having the resistance to heat or cold shock such as E. coli having the resistance to heat or cold shock, E. coli having the improvement of growth, and E. coli having the resistance to osmotic pressure.
- a zinc finger domain is a DNA binding motif of DNA binding proteins that are most frequently discovered in eukaryotes, which is discovered in various species from yeast to higher plant life and human beings.
- the zinc finger domain has been known to function as a transcription repressor that down-regulate the gene expression by itself. Therefore, if fusing the zinc finger domain with the effector domain (up- or down-regulation), the resulted fusing protein, which is a novel transcription factor, may up- or down-regulate the expression of a target gene that is recognized by the zinc finger.
- the zinc finger domain may be a Cys2-His2 type, which three or more zinc finger domains are arranged in parallel to constitute a zing finger protein. Because a single zinc finger domain can recognize a target sequence comprising three or four bases, it can prepare a sequence-specific zinc finger that can selectively recognize target sequences of 9-10 bases by properly re-arranging and linking several zinc finger domains each other.
- one to three zinc finger domains of human genome are arranged to be used as a DNA binding domain of newly developed transcription factors of the present invention.
- the zinc finger domains of the present invention are identified from the human genome.
- it is prepared and used an experimental zinc finger protein that can recognize 5′-GCG GCG GGG-3′ sequence on a reporter plasmid as a target.
- experimental transcription factors comprising effector domain and three zinc finger proteins (ZFP2, ZFP1, and ZFP1, ordered in the N- to C-terminal direction) were prepared by using ZFP1 recognizing 5′-GCG-3′ and ZFP2 recognizing 5′-GGG-3′ as zinc fingers, and used.
- ZFP2, ZFP1, and ZFP1 ordered in the N- to C-terminal direction
- the transcription factors of prokaryotes simultaneously have two functions of activating and repressing transcription. It depends on a position on a genomic sequence in which transcription factors are bound whether transcription is activated or repressed. In general, it is known that when the transcription factors are bound to a portion in range of ⁇ 80 to ⁇ 30 starting from a transcription start point, the gene expression is activated, and when the transcription factors are bound to a portion lower than ⁇ 30 starting from a transcription start point, the gene expression is repressed.
- the present invention is directed to development of transcription factors to be operable in Escherichia coli , the prokaryote. While in case of the eukaryotes different domains are needed for each of transcription activation and repression, in case of the transcription factors of the present invention it may be simultaneously accomplished two functions of activating and repressing transcription by using a single effector domain with good activity.
- transcription activating domain within the transcription factors that substantially repress or activate the gene expression as well as the wild type transcription factors can be used as the effector domain.
- the transcription factors of the procaryotic cell may be used as the effector domain.
- the CRP catabolite regulatory protein, cyclic AMP receptor protein
- the CRP is widely known as a transcription factor that regulates gene expression at over 100 promoters in the Escherichia coli .
- the CRP comprises 209 amino acids and consists of two domains. Namely, the CRP includes an N-terminal domain that is responsible for interaction between dimerization of the CRP and c-AMP and a C-terminal domain that is responsible for interaction with DNA.
- the CRP includes three types of activating domains for transcription activation, such as AR1 (residue 156-164), AR2 (residue 19, 21, 96 and 101), and AR3 (residue 52, 53, 54, 55, 58).
- AR1 is the most favored activating domain in the transcription activation (Busby, S., Ebright, R. H., J Mol Biol, 293, 1999, 199-213; Rhodius, V. A., West, D. M., Webster, C. L., Busby, S. J., Savery N., J. Nucleic Acids Res., 25, 326-332, 1997; Rhodius, V. A., Busby, S. J., J. Mol. Biol., 299, 295-310, 2000; Wagner, R., Transcription regulation in prokaryotes., 199-207 and 211-217. Oxford University Press, Oxford, 2000).
- the zinc finger domain is used as the zinc finger domain
- the CRP derivatives may be used as the effector domain, wherein the CRP derivatives including the CRP being removed the DNA binding domain or comprising the AR1 region.
- the three following types can be used as the effector domain: CRP W (residue 1-209) as the wild-type CRP, CRP Del 137 (residue 137-190), and CRP Del 180 (residue 1-180) as the CRP derivative.
- linkers may be used to link the DNA binding domains to each other or link the DNA binding domains with the effector domain.
- it may use a linker that links the zinc finger domains in naturally occurring zinc finger proteins.
- a typically naturally occurring linker is Thr-Gly-(Glu-Gln)-(Lys-Arg)-Pro-(Tyr-Phe) (SEQ ID NO:65).
- Thr-Gly-Glu-Lys-Pro-Tyr SEQ ID NO:66
- the present invention is prepared for two types of reporter plasmids in order to test the gene expression capability of the artificial transcription factor, wherein the two types of reporter plasmids are pEGFP-A for testing the activation of the gene expression and pEGFP-R for testing the repression of gene expression.
- Each of reporter plasmid were modified by using tac promoter controlled by lacl which is endogenous transcription factor in E. coli in order to test whether or not the transcription factors of the present invention affect. Said modification of tac promoter was introduced by inserting a sequence of 5′-GCG GCG GGG-3,′ which the transcription factor of the present invention may target and bind, into adjacent region of tac promoter in each of reporter plasmid.
- said reporter plasmids were prepared by inserting binding sequence of the test transcription factor into proper region starting from the transcription start point because as aforementioned, it depends on binding position starting from the transcription start point whether the prokaryotic transcription factor activates or represses the gene expression.
- the reporter plasmid in order to demonstrate the gene expression activation capability of test transcription factor, is prepared by means of inserting two copies of 5′-GCG GCG GGG-3′ into a region in range of ⁇ 67 to ⁇ 50 starting from the transcription start point. In order to demonstrate the gene expression repression capability of test transcription factor, the reporter plasmid is prepared by means of inserting two copies of 5′-GCG GCG GGG-3′ into a region in range of +24 to +50 starting from the transcription start point as shown in FIG. 3 .
- GFP derivatives (Clontech Laboratories, Inc., Calif.) may be used as the reporter gene, because they have improved green fluorescent characteristics that facilitate identification and quantitativeness of the gene expression.
- the expression of the GFP derivatives can be detected by measuring fluorescent emission upon being excited with confocal microscopy or spectrofluorometer.
- the present invention relates to artificial transcription factors comprising zinc finger domain and catabolite regulatory proteins derived from the prokaryote used as the effector domain.
- the zinc finger domain of the present invention derives from the eukaryote, it can be also activated in the prokaryote.
- the artificial transcription factors of the present invention can regulate activation of various prokaryotes because it has the catabolite regulatory proteins derived from various prokaryotes as an effector domain.
- the transcription factors of the present invention are introduced, the gene expression can be activated or repressed regardless operation of the endogenous transcription factors in E. coli . Such fact can be used for inducing various E.
- the present invention can be used in various industries.
- FIG. 1 is a scheme showing phenotypic engineering of E. coli by using an artificial transcription factor, in which circles represent zing finger domains and squares represent effector domains;
- FIG. 2 is a scheme showing zing finger proteins used for test transcription factors which are prepared for demonstrating a function of catabolite regulatory protein (or cyclic AMP receptor protein (CRP)) of E. coli and their sequence, in which sequences recognized by the respective zinc finger proteins are shown below the zinc fingers.
- catabolite regulatory protein or cyclic AMP receptor protein (CRP)
- FIGS. 3 and 4 illustrate fragments of reporter plasmids which are prepared by inserting specific sequence recognized by zinc finger protein of test transcription factor in FIG. 2 into up or down stream of tac promoter in order to demonstrate the function of the CRP as the effector domains of the artificial transcription factor, wherein FIG. 3 shows a reporter plasmid pEGPF-A for demonstrating gene expression activation capability of the artificial transcription factors and FIG. 4 shows a reporter plasmid pEGFP-R for demonstrating gene expression repression capability of the artificial transcription factors;
- FIG. 5 shows photos illustrating that the test transcription factor activates the transcription of GFP derivatives (EGFP) reporter gene having improved green fluorescent characteristics of the pEGFP-A, as obtained by a confocal microscopy;
- EGFP GFP derivatives
- FIG. 6 is a graph showing numerically activation degrees of the activated reporter gene expression by the test transcription factors in FIG. 5 ;
- FIG. 7 is a graph showing numerically activation degrees of the repressed reporter gene by the experimental transcription factors in FIG. 5 ;
- FIG. 9 shows photos of E. coli having the resistance to heat shock, which is selected by the artificial transcription factors prepared by using CRP Del 180 as an effector domain;
- FIG. 10 is a graph showing a growth curve of T2, which is an E. coli having the highest resistance to heat shock, at 50° C.;
- FIG. 11 is a graph showing a growth curve of Escherichia coli , which grows better through the artificial transcription factors prepared by using CRP Del 180 as the effector domain, at 37° C. and LB medium;
- FIG. 12 is a graph showing a growth curve of Escherichia coli , which grows better through the artificial transcription factors prepared by using CRP Del 180 as the effector domain, at 37° C. and M9 medium;
- FIG. 13 shows photos of E. coli having the resistance to cold shock, which is selected by the artificial transcription factors prepared by using CRIP Del 180 as the effector domain;
- FIG. 14 is a graph showing a growth curve of CTI, the E. coli having the highest resistance to cold shock, and that of a control;
- FIG. 15 show photos of E. coli having the resistance to osmotic pressure, which is selected by the artificial transcription factors prepared by using CRP Del 180 as the effector domain;
- test zinc finger domains that can recognize 5′-GCG GCG GGG-3′ sequence on a reporter plasmid as a target were prepared by using the following zinc finger domain: ZFP1 recognizing 5′-GCG-3′ and ZFP2 recognizing 5′-GGG-3′.
- CRP W sequence No. 1
- CRP derivative CRP Del 137 residue 137-190, sequence No. 3
- CRP Del 180 residue 1-180, sequence No. 5
- ZFP1 the zinc fingers of the test transcription factors
- ZFP2 the zinc filter of the experimental transcription factors was also selected from the human genome sequence and encoded by human nucleic acid sequence of SEQ ID NO. 11.
- Reporter plasmids which are used for confirmation of activating or repressing the gene expression, were prepared by modifying pACYC184 (New England Biolabs, Inc., USA as shown in FIG. 16 ).
- the reporter plasmids were prepared by amplifying GFP derivative (EGFP) gene having improved green fluorescent characteristics as a reporter gene, a tac promoter gene, and a target sequence of a test transcription regulatory factor with PCR and inserting them to pACYC184.
- the pEGFP-A the reporter plasmid for observing gene expression activation, was prepared by inserting two copies of 5′-GCG GCG GGG-3′ to a region in range from ⁇ 67 to ⁇ 50 starting from a transcription start point (see FIG. 3 ).
- the promoter of pEGFP-R as the reporter plasmid for observing gene expression repression capabilities of the test transcription factors was prepared by inserting two copies of 5′-GCG GCG GGG-3′, the target sequence of the test transcription factors into a region in range from +24 to +41 starting from the transcription start point (see FIG. 4 ).
- SEQ ID NO. 7 is the nucleic acid sequence of the pEGFP-A reporter plasmid
- SEQ ID NO.8 is the nucleic acid sequence of the pEGFP-R reporter plasmid.
- test transcription factors i.e. ZFP-CRP W, ZFP-CRP Del 180 and ZFP-CRP Del 137 prepared in Embodiment 1
- said reporter plasmids were prepared in Embodiment 2
- test transcription factors when adding 1 mM IPTG (Isopropyl- ⁇ -D-thiogalactopyranoside) or not.
- IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
- an activation degree or a repression degree of the reporter gene expression by the test transcription factors according to the present invention was obtained. Specifically, in order to obtain the activation degree, an amount of reporter gene expression in a cell in which a plasmid that encodes the test transcription factors had been introduced was divided by an amount of reporter gene expression obtained from cell in which the control plasmid that does not encode the test transcription factors prepared in embodiment 1 were introduced. Meanwhile, in order to obtain the repression degree, an amount of reporter gene expression obtained from cell in which the control plasmid that does not encode the test transcription factors prepared in embodiment 1 were introduced was divided by an amount of reporter gene expression in a cell in which a plasmid that encodes the test transcription factors had been introduced.
- FIG. 5 The above experimental results are as shown in FIG. 5 .
- ‘No vector’ in FIG. 5 means wild type E. coli in which the reporter plasmid and the plasmid encoding the test transcription factors have not been introduced, and ‘empty vector’ means an E. coli in which the reporter plasmid and the plasmid noncoding the test transcription factors.
- ZFP-CRP W refers to an E. coli in which the reporter plasmid and a plasmid encoding the test transcription factors fused with the wild type CRP as the effector domain are introduced
- ZFP-CRP Del 180 refers to an E.
- test transcription factors according to the present invention activate the expression of the reporter genes of pEGFP-A which had been repressed by not adding IPTG, and that ZFP-CRP W, ZFP-CRP Del 180 and ZFP-CRP Del 137 activated the expression of the EGFP by twice, three and four times, respectively (see FIGS. 5F to 5J and FIG. 7 ).
- 26 types of zinc finger domains of human genomes selected from GenBank database search results were synthesized and cloned into pUC19 (New England Biolabs. Inc., USA, Refer to FIG. 17 ).
- Two different transcription factor libraries were prepared by cloning and fusing CRP Del 137 and CRP Del 180 as the effector domain into transcription factor expression plasmid, respectively.
- the preparation of the transcription factor libraries were made in the same manner as the method for preparation of the experimental transcription factor. This is, the expression plasmid in which the effector domains were cloned was cut with AgeI and EcoRI.
- pUC19 in which different zinc finger domains were cloned were mixed by the same amount and cut with XmaI and EcoRI.
- the expression plasmid cloning the effector domain which was cut with AgeI and EcoRI and the zinc finger domains which was cut with XmaI and EcoRI were ligated to prepare a library comprising the effector domain and a single zinc finger domain. Thereafter, in the same manner above, the expression plasmid in which the effector domain and a single zinc finger domain were cloned was cut with AgeI and EcoRI and then ligated with the zinc finger domain which was cut with XmaI and EcoRI to connect the second zinc finger domain. And finally, the transcription factor library comprising the three zinc finger domains and the effector domain were prepared in the same manner above.
- Z1 to Z26 selected from the human genomes sequences resulted from searching the GenBank database:
- Z1 is encoded by nucleic acid sequence of SEQ ID No. 13;
- Z2 is encoded by nucleic acid sequence of SEQ ID No. 15;
- Z3 is encoded by nucleic acid sequence of SEQ ID No. 17;
- Z4 is encoded by nucleic acid sequence of SEQ ID No. 19;
- Z6 is encoded by nucleic acid sequence of SEQ ID No. 23;
- Z7 is encoded by nucleic acid sequence of SEQ ID No. 25;
- Z8 is encoded by nucleic acid sequence of SEQ ID No. 27;
- Z9 is encoded by nucleic acid sequence of SEQ ID No. 29;
- Z10 is encoded by nucleic acid sequence of SEQ ID No. 31;
- Z11 is encoded by nucleic acid sequence of SEQ ID No. 33;
- Z12 is encoded by nucleic acid sequence of SEQ ID No. 35;
- Z13 is encoded by nucleic acid sequence of SEQ ID No. 37;
- Z14 is encoded by nucleic acid sequence of SEQ ID No. 39;
- Z15 is encoded by nucleic acid sequence of SEQ ID No. 41;
- Z16 is encoded by nucleic acid sequence of SEQ ID No. 43;
- Z17 is encoded by nucleic acid sequence of SEQ ID No. 45;
- Z18 is encoded by nucleic acid sequence of SEQ ID No. 47;
- Z19 is encoded by nucleic acid sequence of SEQ ID No. 49;
- Z20 is encoded by nucleic acid sequence of SEQ ID No. 51;
- Z21 is encoded by nucleic acid sequence of SEQ ID No. 53;
- Z22 is encoded by nucleic acid sequence of SEQ ID No. 55;
- Z23 is encoded by nucleic acid sequence of SEQ ID No. 57;
- Z24 is encoded by nucleic acid sequence of SEQ ID No. 59;
- Z25 is encoded by nucleic acid sequence of SEQ ID No. 61;
- Z26 is encoded by nucleic acid sequence of SEQ ID No. 63.
- E. coli having desired phenotypes under particular condition was induced and selected by introducing the transcription factor library prepared in experimental example 2 into E. coli .
- Transcription factors were isolated from the E. coli having the desired phenotypes, and re-transformed to the E. coli to confirm whether the same phenotypes were induced to thus confirm the induction of the particular phenotypes by the transcription factors.
- an E. coli having artificial transcription factors comprising CRP Del 180 as the effector domain was thermally shocked during two hours at 55° C., and plated on an LB plate, and then cultivated at 37° C. After that, an E. coli having heat resistance was selected from colonies which had been grown from the cultivation.
- Transcription factors were isolated from the selected E. coli and confirmed through DNA sequence analysis, which were then re-transformed into the E. coli in order to confirm whether their heat resistance was induced, thus confirming activity of the transcription factors. Observing a growth pattern of the E. coli at 100 ml LB medium at 50° C., samples of the E. coli were taken at intervals of about one hour and half minutes to measure optical density (OD) at spectrophotometer 600 nm to obtain a growth curve of the Escherichia coli.
- OD optical density
- FIG. 9 shows the results of the experimentation.
- the left photo in FIG. 9 is a photo of a control of the E. coli to which heat shock was not applied, while the right photo shows a survivality of the E. coli after heat shock was applied for 2 hours at 55° C.
- ‘C’ in the photos indicates control E. coli having control plasmid in which artificial transcription factors were not encoded, while T1 to T3 indicate E. coli having heat resistance by different artificial transcription factors.
- FIG. 9 under the conditions in which no heat shock was applied, every E. coli formed colonies, while under the conditions in which the heat shock was applied, growth of the wild-type E. coli was hampered but the E. coli having the transcription factors comprising CRP Del 180 as the effector domains according to the present invention grew well even with the heat shock.
- the triangles at upper portions in FIG. 9 indicate a 5 ⁇ diluted cell density.
- CRP Del 180 is used as the effector domain, and the transcription factor library prepared in experimentation example 2 was introduced to induce E. coli having the improved growth rate at 37° C., an optimal growth temperature and be screened.
- the transcription factor library After introducing the transcription factor library to the Escherichia coli , it was subcultured (transferred 1 ml by 1 ml to a new 100 ml medium at intervals of 24 hours during seven days) at 37° C. at the LB medium and M9 medium of 100 ml, streaked or diluted, and then plated to obtain colonies. A growth pattern of each colony was checked at the LB medium and M9 medium of 100 ml at 37° C. and E. coli having improved growth rate was finally selected. Transcription factors of the selected E. coli was isolated and checked if they are transcription factors through DNA sequence analysis, which is then re-transformed into E. coli to confirm whether the growth rate has been improved, thereby confirming the improvement of the growth rate by the transcription factors.
- FIGS. 11 and 12 show the results obtained by checking the growth rate at the LB medium at 37° C. and FIG. 12 shows the results obtained by checking the growth speed at M9 medium at 37° C.
- C indicates control E. coli having the control plasmid in which the artificial transcription factors were not encoded
- RG#32 indicates E. coli having improved growth rate by the artificial transcription factors.
- FIG. 11 in case of the E. coli in which the artificial transcription factors were not encoded, its growth rate was not increased after 15 hours of its cultivation, while in case of the E. coli having the transcription factors according to the present invention, it were grown continuously until 50 hours after its cultivation. Accordingly, it have been demonstrated that the productivity of the E. coli was remarkably improved.
- RG#32 CRP1-180 a.a+Z 26 +Z 7
- the zinc finger proteins, the DNA binding domains of the artificial transcription factors in the present invention can have activation in the prokaryote as well as in the eukaryote.
- the CRP used as the effector domain of the artificial transcription factors has the same sequence with that of CRP of Shigella and its family proteins exist in various prokaryotes, it has been estimated that the artificial transcription factors according to the present invention can regulate the gene expression of various prokaryotes as well as E. coli and induce various phenotypes thereof.
- an E. coli having the resistance to a low temperature may be grown well, which have a high value as an industrial microorganism. Therefore, in order to induce a E. coli having resistance to cold shock, the following experimentation was performed. In this experimentation, CRP Del 180 was used as the effector domain, and the transcription factor library prepared in the experimentation example 2 was introduced to induce an E. coli that can be grown at a low temperature and be screened.
- CT 1 Cp 1 ⁇ 180 a.a+Z 19 +Z 6 +Z 22
- CT 2 CP 1 ⁇ 180 a.a+Z 15 +Z 4 +Z 2
- coli having resistance to osmotic pressure was selected from the grown colonies. Transcription factors were isolated from the selected Escherichia coli , checked through the DNA sequence analysis, and re-transformed into E. coli in order to check whether resistance to the osmotic pressure was induced, thereby confirming activity of the transcription factors.
- FIG. 15 shows the results of the experimentation.
- the left photo in FIG. 15 is a photo of a control of the E. coli grown in a minimum A medium to which osmotic pressure was not given, while the right photo shows a survivality of the E. coli in a minimum A medium to which osmotic pressure was given.
- ‘C’ in the photos indicates control E. coli having control plasmid in which artificial transcription factors were not encoded, while OT1, OT2, OT3 and OT4 indicate E. coli in which different artificial transcription factors were introduced, having resistance to osmotic pressure.
- the triangles at upper portions in FIG. 15 indicate a ⁇ 5 diluted cell density.
- OT 1 CPP 1 ⁇ 180 a.a+Z 24+ Z 2+ Z 2
- OT 2 CP 1 ⁇ 180 a.a+Z 8+ Z 4+ Z 19
- OT 3 CRP 1 ⁇ 180 a.a+Z 5+ Z 4+ Z 17
- OT 4 CRP 1 ⁇ 180 a.a+Z 9+ Z 24+ Z 23
- E. coli having the desired phenotypes can be induced, such as E. coli having the resistance to heat or cold shock, or osmotic pressure, or improvement of growth rate.
Abstract
Description
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300602006100Seqlist.txt | 22 Jan. 2010 | 37,773 bytes |
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EP2084180A4 (en) | 2010-04-21 |
KR100812110B1 (en) | 2008-03-12 |
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